Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch.

Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3'UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ required five upstream adenylates, and H4a/Ψ was necessary for cooperative association of two other hairpins (H5/H4b) in Mg. SMD recapitulated the TSS unfolding order in the absence of Mg, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3'UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.